Anti srebp 1c

Total protein was extracted from isolated zebrafish samples, using a lysis buffer (0.5% NP-40, 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1 mM EDTA). Protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Hepatic protein extract (20–50 μg) was separated by 12% sodium dodecyl sulphate–polyacrylamide electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked in 5% non-fat dried milk in phosphate-buffered saline 0.05% (v/v) and Tween 20 (PBST; pH 7.4) (Sigma-Aldrich, St. Louis, MO) and probed with antibodies that had been diluted at 1:500–1000 in a 0.3% solution of bovine serum albumin in PBST. Anti-V5 antibody was purchased from Invitrogen. Anti-PPARγ, anti-C/EBP-β, and anti- SREBP-1c antibodies were purchased from Santa Cruz. β-actin served as the loading control and detected using anti-β-actin antibody (Sigma-Aldrich) diluted at 1:1000 in PBST.

The cytoplasmic proteins of liver tissues and hepatocytes were extracted according to the instructions of the cytoplasmic protein extraction kit (Beyotime, Beijing, China). A total of 30–60 μg cytoplasmic protein was resolved on an 8%–12% precast gel using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Invitrogen, NY, USA) and transferred to polyvinylidene fluoride membranes (Bio-Rad, CA, USA). The membranes were then incubated with anti-p-AMPK, anti-AMPK, anti-G6Pase, anti-PEPCK, anti-ACC, anti-p-ACC, anti-FAS, anti-CPT-1a, anti-SHP1, anti-SREBP-1c (Santa Cruz, CA, USA), and anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Cell Signaling, MA, USA). Then, these membranes were washed with PBS and incubated with anti-mouse or anti-rabbit secondary antibody (Beyotime) for 1 h at room temperature. Finally, the immune complexes were developed using an enhanced chemiluminescence western blotting substrate, and protein expression levels were quantified using ImageJ software.